Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.
作者： Penporn Sujiwattanarat , Parinya Pongsanarakul , Yosapong Temsiripong , Theeranan Temsiripong , Charin Thawornkuno
DOI: 10.1016/J.CBPA.2015.10.028
关键词: Superoxide dismutase 、 Peptide sequence 、 Protein purification 、 Amino acid 、 Crocodylus siamensis 、 Biology 、 Molecular cloning 、 Gene 、 Gene expression 、 Molecular biology 、 Biochemistry
摘要: Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein 154 amino acids without signal peptides, indicated intracellular CSI-Cu,Zn-SOD. This agreed with the results phylogenetic tree, which that CSI-Cu,Zn-SOD belonged Cu,Zn-SOD. Chromosomal location determined localized proximal region chromosome 1p. Several highly conserved motifs, two signature sequences (GFHVHEFGDNT GNAGGRLACGVI), acid residues binding copper zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), His(120)) were also identified Real-time PCR analysis showed mRNA tissues examined (liver, pancreas, lung, kidney, heart, whole blood), suggests constitutively these tissues. Expression Escherichia coli cells followed purification yielded recombinant CSI-Cu,Zn-SOD, Km Vmax values 6.075mM xanthine 1.4×10(-3)mmolmin(-1)mg(-1), respectively. value 40 times lower than native (56×10(-3)mmolmin(-1)mg(-1)), extracted erythrocytes. cofactors, folding properties, or post-translational modifications lost during process, leading reduction rate activity bacterial expression
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